Simultaneous determination of danshensu,rosmarinic acid,cryptotanshinone, tanshinone IIA,tanshinone I and dihydrotanshinone I by liquid chromatographic–mass spectrometry and the application to pharmacokinetics in rats

A sensitive and specific liquid chromatography–tandem mass spectrometry method (LC–MS) was developed and validated for the separation and simultaneous determination of danshensu, rosmarinic acid and tanshinone compounds including cryptotanshinone, tanshinone I, dihydrotanshinone I and tanshinone IIA in rat plasma. Chromatographic separation of the analytes was successfully achieved on a C₁₈ column using a mobile phase composed of acetonitrile–water containing 0.5% glacial acetic acid. This method demonstrated good linearity and did not have endogenous material interfering with the active compounds and I.S. peaks. The limit of quantification of danshensu, rosmarinic acid, cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA were 5, 0.75, 0.1, 0.1, 1 and 0.5 ng/mL. The average extraction recoveries of these analytes from rat plasma were all over 60%. The precisions determined from five days were all within 10%. This method has been successfully applied in the simultaneous quantification and the pharmacokinetic studies of these six compounds in animals which were orally administered with danshen preparations.     

Simultaneous determination of danshensu, ferulic acid, cryptotanshinone and tanshinone IIA in rabbit plasma by HPLC and their pharmacokinetic application in danxiongfang

A selective and sensitive reversed-phase high performance liquid chromatography method was developed and validated for the simultaneous determination of danshensu, ferulic acid, cryptotanshinone, and tanshinone IIA in rabbit plasma using p-hydroxybenzoic acid as internal standard. Liquid–liquid extraction was used for sample preparation. Chromatographic separation was successfully achieved on an Agilent HC-C₁₈ column using a mobile phase composed of methanol–water (from 20:80 to 80:20, v/v) containing 0.5% (v/v) glacial acetic acid. The mobile phase was employing gradient elution at a flow rate of 1.0 ml/min. The method showed good linearity and no endogenous material interfered with the marked compounds and I.S. peaks. The limit of quantification of danshensu, ferulic acid, cryptotanshinone, and tanshinone IIA were 0.1, 0.03, 0.05, and 0.05 μg/ml, respectively. The average extract recoveries of the four compounds from rabbit plasma were all over 60%. The precisions determined from 5 days were all within 10%. The established method has been successfully applied in the pharmacokinetic study and drug interaction of danshensu, ferulic acid, cryptotanshinone, and tanshinone IIA in rabbits after intravenous administration of danxiongfang, a useful compound preparation of traditional Chinese medicine.     

Improved quality control method for Danshen products--consideration of both hydrophilic and lipophilic active components

The current study intends to provide an improved quality control analysis for Danshen product-a representative herbal product with known active components that are both hydrophilic and lipophilic in nature. A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of three major lipophilic components (cryptotanshinone, tanshinone I and tanshinone IIA) and three major hydrophilic components (danshensu, protocatechuic aldehyde and salvianolic acid B) of Danshen (Salvia miltiorrhiza). These six components were successfully separated using Radial-pak C18 cartridge with the elution gradient consisting of 0.5% acetic acid in water and 0.5% acetic acid in acetonitrile at a flow rate of 1 ml/min. The intra-day and inter-day precisions of the analysis were within 2.32 and 2.0%, respectively. The detection limits were 0.02, 0.01, 0.01, 0.05, 0.005 and 0.02 microg/ml for cryptotanshinone, tanshinone I, tanshinone IIA, danshensu, protocatechuic aldehyde and salvianolic acid B, respectively. The developed method has been applied to the simultaneous determination of above six major components in Fufang Danshen Tablet and Dripping Pill products by extraction with methanol and water. It has been demonstrated that salvianolic acid B and danshensu are the major components among the eight commercial Fufang Danshen products studied. The current developed method with methanol as extraction solvent provides a simple and efficient method for simultaneous detection of both lipophilic and hydrophilic major components in Danshen products.     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.     

Application of non-aqueous micellar electrokinetic chromatography to the analysis of active components in radix Salviae miltiorrhizae and its medicinal preparations

A simple, economical and effective non-aqueous micellar electrokinetic chromatography (NAMEKC) method was developed for simultaneous assay of three bioactive components (cryptotanshinone, tanshinone IIA and tanshinone I) in radix Salviae miltiorrhizae and its medicinal preparations for the first time. After optimization of separation conditions, a buffer of 140 mmol l(-1) sodium cholate (SC) in methanol was selected for the separation of the three tanshinones, but baseline separation of tanshinone I and tanshinone IIA in practical samples was not achieved. Therefore, second-order derivative electropherograms were applied for resolving overlapping peaks. Regression equations revealed good linear relationships (correlation coefficients 0.995-0.999) between peak heights in second-order derivative electropherograms and concentrations of the three analytes. The recoveries of three constituents ranged from 91.3 to 105.7%. The results indicated that baseline separation of the analytes was hard to be achieved in practical samples sometimes and second-order derivative electropherograms was applicable for the resolving and analysis of overlapping peaks.     

Simultaneous determination of cryptotanshinone, tanshinone I and tanshinone IIA in traditional Chinese medicinal preparations containing Radix salvia miltiorrhiza by HPLC

A reversed-phase high performance liquid chromatographic method was established for the simultaneous determination of tanshinones in five kinds of traditional Chinese medicinal preparations (TCMPs) containing Radix salvia miltiorrhiza (Chinese herbal name: Danshen). Tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA were successfully separated on a Diamonsil C18 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of methanol, tetrahydrofuran, water and glacial acetic acid (20:35:44:1, v/v/v/v), employing isocratic elution at a flow rate of 1.0 mL/min. Detection was accomplished at 254 nm. The compounds were identified by comparing their retention times and UV spectra in the 200-400 nm range with authentic standards. Regression equations revealed good linear relationship between the peak areas of the constituents and their concentrations (correlation coefficients: 0.9998 for cryptotanshinone, 0.9999 for tanshinone I and 1.0000 for tanshinone IIA). The relative standard deviations (n=6) of retention time and peak area were less than 0.25% and 1.00%, respectively. The recoveries were between 96.2% and 102.5%. The proposed method has been successfully applied to the simultaneous determination of the tanshinones in five kinds of Chinese herbal preparations containing Danshen within 20 min.     

Cytotoxicity of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on HepG2 cells in relation to glutathione perturbation.

Tanshinones are abietane type-diterpene quinones isolated from the roots of Radix Salvia miltiorrhiza (Danshen), a well-known traditional Chinese medicine in the treatment of cardiovascular diseases. Among the major diterpenes isolated, including cryptotanshinone, tanshinone I, tanshinone IIA and dihydrotanshinone, tanshinone IIA had been shown to posses various pharmacological activities including antioxidant, protection/prevention from angina pectoris and myocardial infarction, and anticancer properties. Tanshinone IIA, usually the most abundant tanshinone present in the herb, has been the focus of studies in its clinical potential, among which its ability to inhibit the proliferation of cancer cell lines. The aim of this study was to study the cytotoxicity of the tanshinones on human HepG2 cells in vitro in relation to intracellular glutathione perturbation (reduced glutathione, GSH and oxidized glutathione, GSSG). Studies using MTT assay showed that all tanshinones decreased cell viability of HepG2 cells in a concentration-dependent manner, with the cell viability decreased to 60% and 35% after 24 h and 48 h treatment, respectively. Assessment of apoptotic cells with fragmented DNA by flow cytometry indicated that only tanshinone IIA (12.5 and 25 microM) induced apoptosis in the cancer cells. Tanshinone IIA and cryptotanshinone caused significant decreases in G(1) cells by 23% and 13%, respectively, after 24 h treatment. The declines in G(1) cells were compensated by increases in G(2)/M (15% for tanshinone IIA) and S cells (8% and 13% for tanshinone IIA and cryptotanshinone, respectively). All the tanshinones studied, except tanshinone IIA, elevated GSH/GSSG ratio at low concentrations (1.56 and 3.13 microM), but the ratio decreased, indicating oxidative stress at high concentrations (6.25-25 microM). Taken together, tanshinone IIA caused HepG2 cytotoxicity through apoptosis without influencing oxidative stress, while the other tanshinones showed lower efficacy in inducing apoptosis in the HepG2 cells.     

Quantitative determination of pravastatin and its biotransformation products in human serum by turbo ion spray LC/MS/MS

A sensitive, specific, accurate and reproducible analytical method was developed and validated to quantify pravastatin (Prav), pravastatin-d5 (Prav-d5), SQ-31906, SQ-31906-d5, and pravastatin lactone (Prav-Lac) in human serum samples. Serum samples (0.5 ml) were acidified and extracted by a solid-phase extraction procedure to isolate all five analytes from human serum. Sample extracts were reconstituted and analyzed by turbo ion spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the positive ion mode. The total run time was 9 min between injections. The assay demonstrated a lower limit of quantitation (LLQ) of 0.5 ng/ml for all five analytes. The calibration curves were linear from 0.5 ng ml to 100 ng/ml for all five analytes. The coefficients of determination of all calibration curves were > or = 0.999. Precision and accuracy quality control (QC) samples were prepared at concentrations of 2, 30, 80, and 500 ng/ml for all analytes. The intra-assay and inter-assay precision calculated from QC samples were within 8%, for all analytes. The inter-assay accuracy calculated from QC samples was within 8% for all analytes. The extraction recoveries were > or = 90% for all analytes. Benchtop stability experiments in an ice-water bath ( < or = 10 degrees C) demonstrated that over time, Prav-Lac hydrolyzes to Prav in serum. Prav, Prav-d5, SQ-31906, and SQ-31906-d5 were stable under these conditions for up to 24 h. Hydrolysis was minimized by buffering the serum to pH 4.5 and maintaining the serum sample in an ice-water bath. All analytes were stable after three freeze/thaw cycles and in reconstitution solution after 1 week at 4 degrees C. Stability of all analytes in human serum was demonstrated after storage at -70 degrees C for 77 days. The benchtop (< or = 10 degrees C) stability of pooled study samples was also investigated and the results were comparable to those obtained from serum QC samples.     

Enhancement of solubility and dissolution rate of cryptotanshinone, tanshinone I and tanshinone IIA extracted from Salvia miltiorrhiza

Cryptotanshinone, tanshinone I and tanshinone IIA are three major components in the extract of Salvia miltiorrhiza with pharmacological significance. However, their effective utilization is limited due to poor water solubility and bioavailability. Solid dispersion (SD) of the extract of Salvia miltiorrhiza was prepared to enhance solubility and dissolution of the three major components. Various carriers were screened for SD preparation by conventional solvent method. Dissolution of the components from selected SD systems was compared with commercial tablets of the extract from Salvia miltiorrhiza. The solubility of three components viz., cryptotanshinone, tanshinone I and tanshinone IIA, after forming SD with either of povidone K-30 (PVP K-30) or poloxamer 407, exhibited enhanced solubility in pH 6.8 buffer. Dissolution test revealed that the amount of three components released was higher from SD tablets as compared to the commercial tablets. Pharmacokinetic profile was evaluated using cryptotanshinone as a representative compound. AUC of cryptotanshinone was significantly increased when administered as a solid dispersion.     

Study of sodium tanshinone II A sulfonate tissue distribution in rat by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and fully validated for the quantitative determination of sodium tanshinone IIA sulfonate (STS, sodium (1,6,6-trimethyl-10,11-dioxo-7,8,9-trihydrophenanthro[1,2-b]furan)-yl-2-sulfonate) in rat biosamples including plasma and different tissues using sodium tanshinone I sulfonate (sodium (1,6-dimethyl-10,11-dioxo-phenanthro[1,2-b]furan)-yl-2-sulfonate) as internal standard. Simple protein precipitation by acetonitrile was utilized for extracting STS from the rat biosamples. Chromatographic separation of the sample matrix from the analyte and the internal standard was performed using a commercially available analytical column with a mobile phase consisting of methanol-5 mmoL/L ammonia acetate (70:30, v/v) at a flow rate of 0.2 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in the negative-ion mode. The intra- and inter-day precisions (RSD%) and deviations of the assay accuracies were within 10.0% for STS. The extraction recovery of STS was more than 86.5%. The limit of detection (LOD) of STS was 1.0 ng/mL. The method was successfully applied to the tissue distribution study of STS intravenously administered to healthy Sprague-Dawley rats. The tissue distribution results showed that liver, kidney, lung, small intestine and duodenum were the major distribution tissues of STS in rats, and that STS had difficulty in crossing the blood-brain barrier. After 24 h, STS could be detected only in the kidney, stomach and small intestine, indicating that there was no long-term accumulation of STS in rat.     

Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen—antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid–liquid extraction with diethyl ether and analytes were chromatographed on a C₈ chromatographic column by elution with acetonitrile–water–formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1–2090 pg/ml for flupentixol and 0.206–4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9–75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15–5.92% with accuracy of 97.6–103.0% for flupentixol and 0.5–6.36% with accuracy of 98.7–101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.     

A UFLC/MS/MS method for simultaneous quantitation of alisol A and alisol B 23-acetate from Alisma orientale (Sam.) Juz. in rat plasma

A sensitive and reliable ultra fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS) method has been developed and validated for simultaneous quantitation of alisol A and alisol B 23-acetate from Alisma orientale (Sam.) Juz. in rat plasma using diazepam as an internal standard (IS). The plasma samples were extracted by liquid–liquid extraction with methyl tert-butyl ether and separated on a Venusil MP C18 column (100 mm × 2.1 mm, 3.0 mm) (Venusil, China) using gradient elution with the mobile phase consisting of methanol and 0.1% acetic acid in water at a flow rate of 0.4 ml/min. The two analytes were monitored with positive electrospray ionization by multiple reaction monitoring mode (MRM). The lower limit of quantitation was 5.00 ng/ml for alisol A and 5.00 ng/ml for alisol B 23-acetate. The calibration curves were linear in the range of 5.00–2500 ng/ml for alisol A and 5–2500 ng/ml for alisol B 23-acetate. The mean extraction recoveries were above 63.8% for alisol A and 68.0% for alisol B 23-acetate from biological matrixes. Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (15%). The validated method was successfully applied to the pharmacokinetic study of alisol A and alisol B 23-acetate in rat plasma after oral administration of alcohol extract of Alismatis Rhizoma.     

PF2401-SF, standardized fraction of Salvia miltiorrhiza and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, protect primary cultured rat hepatocytes from bile acid-induced apoptosis by inhibiting JNK phosphorylation.

Bile acid-induced hepatocyte apoptosis plays an important role in cholestatic liver disease, and the role of apoptosis may be of therapeutic interest in preventing liver disease. The dried root of Salvia miltiorrhiza Bunge (Labiatae) has been used traditionally to treat liver diseases. We investigated the antiapoptotic effects of a standardized fraction of S. miltiorrhiza (PF2401-SF) and its components, tanshinone I, tanshinone IIA, and cryptotanshinone, in primary cultured rat hepatocytes. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). Glycochenodeoxycholic acid (GCDC)-induced apoptosis, as shown by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and activation of caspases-8, -9, and -3. PF2401-SF and its components, tanshinone I, tanshinone IIA, and cryptotanshinone showed antiapoptotic activity. Treatment with PF2401-SF or with its components significantly inhibited the generation of intracellular reactive oxygen species. Hydrophobic bile acids activate c-Jun-NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinase 1/2, and PF2401-SF inhibited the phosphorylation of JNK and p38. All three components of PF2401-SF inhibited JNK phosphorylation. Addition of inhibitors of MAPK showed that inhibition of JNK decreased apoptosis. These data indicate that PF2401-SF and its components protect hepatocytes from GCDC-induced apoptosis in vitro by inhibiting JNK.     

Growth-inhibitory and apoptosis-inducing effects of tanshinones on hematological malignancy cells and their structure-activity relationship

This study has investigated the growth-inhibitory and apoptosis-inducing effects of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone on hematological malignancy cell lines, aiming to explore their structure-activity relationship. The growth-inhibitory effects of the tanshinones on K562 and Raji cells were assessed using a modified MTT assay; the apoptosis-inducing effects were assessed by fluorescence microscopy and flow cytometry analysis. The changes in cellular morphology were observed using an inverted phase-contrast microscope. MTT results revealed that these tanshinones inhibited cell proliferation in a concentration-dependent and time-dependent manner. The IC50 values of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone for K562 cells were 3.50, 13.52, 19.32, and 47.52 μmol/l at 24 h; 1.36, 4.70, 5.67, and 22.72 μmol/l at 48 h; and 1.15, 1.59, 2.82, and 19.53 μmol/l at 72 h, and the values for Raji cells were 3.30, 4.37, 12.92, and 52.36 μmol/l at 24 h; 1.55, 1.71, 6.54, and 25.45 μmol/l at 48 h; and 1.07, 1.38, 1.89, and 18.47 μmol/l at 72 h. The flow cytometry analysis demonstrated that these tanshinones induced apoptosis of K562 cells in a concentration-dependent manner, and dihydrotanshinone as well as tanshinone I were more potent than tanshinone IIA and cryptotanshinone. Some noticeable apoptotic morphologies could be observed by fluorescence microscopy on tanshinones-treated Raji cells. Collectively, these tanshinones caused growth inhibition and apoptosis in hematological malignancy cell lines, with dihydrotanshinone being the most potent, followed by tanshinone I, tanshinone IIA, and cryptotanshinone. These results suggested that the structure of aromatic ring A enhanced the cytotoxicity and the structure of ring C may have contributed to the cytotoxicity, but the mechanisms need to be further investigated.     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

1. This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone.

2. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed.

3. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not.

4. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.

     

Tanshinone congeners improve memory impairments induced by scopolamine on passive avoidance tasks in mice

Tanshinones are a group of diterpenoids found in the roots of Salvia miltiorrhiza Bunge which has been used to treat cardiac disease. In the present study, we investigated the effect of the tanshinone congeners, tanshinone I, tanshinone IIA, cryptotanshinone, and 15, 16-dihydrotanshinone I, on learning and memory impairments induced by scopolamine (1 mg/kg, i.p.), a muscarinic antagonist, using passive avoidance tasks in mice. Tacrine was used as a positive control. Tanshinone I (2 or 4 mg/kg, p.o.), tanshinone IIA (10 or 20 mg/kg, p.o.), cryptotanshinone (10 mg/kg, p.o.), and 15, 16-dihydrotanshinone I (2 or 4 mg/kg, p.o.) significantly reversed scopolamine-induced cognitive impairments (P<0.05). Tanshinone I (2 mg/kg, p.o.) and tanshinone IIA (10 or 20 mg/kg, p.o.) were also reversed diazepam-induced cognitive dysfunctions (P<0.05). In addition, cryptotanshinone and 15, 16-dihydrotanshinone I were found to have an inhibitory effect on acetylcholinesterase in vitro with IC(50) values 82 and 25 microM, respectively. Furthermore, cryptotanshinone inhibited acetylcholinesterase activity for 3 h and 15, 16-dihydrotanshinone I for 6 h in an ex-vivo study. These results suggest that tanshinone congeners may be useful for the treatment of cognitive impairment and that their beneficial effects are mediated, in part, by cholinergic signaling enhancement.     

Effects of the coexisting diterpenoid tanshinones on the pharmacokinetics of cryptotanshinone and tanshinone IIA in rat

Tanshinone II_A and cryptotanshinone are the main pharmacologically active components in the Chinese herb drug Salvia miltiorrhiza Bge. The objective of this study was to investigate the effect of coexisting tanshinones in liposoluble ethanol extract of S. miltiorrhiza Bge. on the rat pharmacokinetics of tanshinone II_A and cryptotanshinone after oral intra-gavage administration of the tanshinones extract. Rats were given the tanshinones extract 23.3 mg/kg (equivalent to 5.7 mg/kg cryptotanshinone and 8.0 mg/kg tanshinone II_A), cryptotanshinone 5.7 mg/kg, or tanshinone II_A 8.0 mg/kg orally under overnight fasted conditions. Blood samples were taken at predetermined sampling time interval and the concentrations of cryptotanshinone and tanshinone II_A were determined by a validated LC–MS/MS method. The peak plasma concentrations of cryptotanshinone and tanshinone II_A were considerably increased (about 8 and 10 folds) after oral administration of the extract in comparison with the equivalent dose of single component administration, respectively. The areas under the plasma concentration–time curve (AUC) of cryptotanshinone and tanshinone II_A were both significantly increased (P < 0.001) as well. Tanshinone II_A was also found after the administration of cryptotanshinone alone, and the fraction of metabolism of tanshinone II_A was 21.0 ± 4.1%. Therefore, the pharmacokinetics of cryptotanshinone and tanshinone II_A in rats after administration of the tanshinones extract were significantly affected by the coexisting tanshinones. In conclusion, the herb-drug interactions occurred between coexisting tanshinones and cryptotanshinone or tanshinone II_A affected their absorption, transformation and metabolism.     

Quantitation of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide and 4-amino-3,5-dichloropyridine in rat and mouse plasma by LC/MS/MS

The metabolism of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (RP73401), a phosphodiesterase IV (PDE IV) inhibitor is extensive (unpublished); however, until recently, studies for this compound did not report 4-amino-3,5-dichloropyridine (ADCP) as a metabolite either in vitro or in vivo. This prompted a reinvestigation into the metabolism of RP73401 in rats and mice using mass spectrometry. The results of the reinvestigation confirmed that 4-amino-3,5-dichloropyridine was formed via the metabolism of RP73401 both in vitro and in vivo. In order to further investigate RP73401 hydrolysis in vivo, a liquid chromatography/mass spectrometry assay was developed and validated for the simultaneous determination of RP73401 and ADCP in rat and mouse plasma. The method used Waters Oasis HLB brand solid phase extraction cartridges to isolate the analytes (RP73401 and ADCP) and internal standard from the plasma. HPLC chromatographic separation was achieved using a Zorbax SB C18 HPLC column and detection was accomplished using positive ion atmospheric pressure chemical ionization tandem mass spectroscopy in multiple reaction monitoring (MRM) mode. The assay was developed and validated over the range of 0.5-100 ng ml(-1) for RP73401 and 5-500 ng ml(-1) for ADCP using 0.050 ml of plasma. The assay proved to be sensitive, accurate, precise and specific for RP73401 and ADCP. Intraday and interday quality control results routinely showed accuracy and precision to be within +/- 20%. This LC/MS/MS method was subsequently employed to investigate the hydrolysis of RP73401 in the rat and mouse, and determine the effects of tri-o-tolyl phosphate (TOTP, a carboxylesterase inhibitor) preadministration on the hydrolysis reaction in the rat.     

Diterpene quinone tanshinone IIA selectively inhibits mouse and human cytochrome P4501A2

1. Tanshinone IIA is the main active diterpene quinone in the herbal medicine Salvia miltiorrhiza. In untreated mouse liver microsomes, tanshinone IIA selectively inhibited 7-ethoxyresorufin O -deethylation (EROD) and 7-methoxyresorufin O -demethylation (MROD) activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, N -nitrosodimethylamine and nifedipine. Tanshinone IIA was a competitive inhibitor of MROD activity with a K i of 7.2 ± 0.7 nM. 2. In 3-methylcholanthrene-treated mouse liver microsomes, tanshinone IIA and two minor tanshinones, tanshinone I and cryptotanshinone, inhibited liver microsomal MROD activity without affecting EROD and benzo(a)pyrene hydroxylation activities at the concentrations up to 1 µ M. Tanshinone IIA induced a type I binding spectrum with a spectral dissociation constant K?s of 2.3 ± 0.8 µ M without cooperativity. 3. In human liver microsomes, tanshinone IIA decreased EROD and MROD activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, chlorzoxazone and nifedipine. 4. In Escherichia coli membranes expressing bicistronic human CYP1A enzymes, tanshinone IIA inhibited EROD activity of CYP1A1 with an IC 50 48 times higher than that for CYP1A2. Tanshinone I and cryptotanshinone had the same IC 50 ratio (1A1/1A2) of 4. 5. The results indicate that tanshinone represents a new group of CYP1A inhibitors, and tanshinone IIA had the highest selectivity in inhibition of CYP1A2.     

Preventive effects of a purified extract isolated from Salvia miltiorrhiza enriched with tanshinone I,tanshinone IIA and cryptotanshinone on hepatocyte injury in vitro and in vivo

Salvia miltiorrhiza is traditionally used to treat liver disease in Asia. In this study, we tested the ability of a purified extract of S. miltiorrhiza (PF2401-SF) and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, to protect against acute and subacute liver damage induced by carbon tetrachloride by measuring serum transaminase levels, the reduced form of glutathione (GSH), antioxidant enzyme activities, and lipid peroxidation levels in the liver. We also evaluated their ability to protect primary cultured rat hepatocytes from tertiary-butylhydroperoxide (tBH) or d-galactosamine (GalN). PF2401-SF was protective at 50–200 mg/kg per day in acute liver injury and 25–100 mg/kg per day in subacute liver injury. Tanshinone I, tanshinone IIA, and cryptotanshinon (40 μM), inhibited lactate dehydrogenase leakage, GSH depletion, lipid peroxidation and free radical generation in vitro. PF2401-SF and its major constituents, tanshinone I, tanshinone IIA and cryptotanshinone, can protect against liver toxicity in vivo and in vitro due to its antioxidant effects.